The plant toxin ricin is one of the most toxic substances known and can cause severe morbidity and mortality. It is a category B select agent. There are no specific protective measures or therapeutics effective against ricin intoxication and there is an urgent unmet need for therapy. Therefore, understanding how ricin kills cells and developing antidotes to protect exposed people remain top health priorities. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved ?-sarcin/ricin loop (SRL) in the large rRNA. The toxicity of ricin is thought to be due to irreversibe inactivation of ribosomes and subsequent translational arrest. Our work challenged this paradigm by demonstrating that ribosome depurination does not directly correlate with the cytotoxicity of RTA in yeast and in mammalian cells. We showed that RTA binds to the ribosomal stalk to depurinate ribosomes with an exceptionally high rate of association and dissociation, allowing it to depurinate the SRL at a much higher rate on intact ribosomes than on the naked 28S rRNA. Our preliminary results in human cells demonstrated that the human ribosomal stalk is also critical for the depurination activity of RTA. We present new preliminary evidence that the ribosome binding surface of RTA, which is distinct from the active site, is required for full toxicity. We showed that RTA inhibits the unfolded protein response (UPR) in yeast and in mammalian cells and inhibition of the UPR contributes to cytotoxicity of ricin. Our genome-wide screen in yeast identified novel host factors that mediate the toxicity of RTA. We obtained recent evidence that N-glycosylation is important for dislocation of RTA from the ER to the cytosol and identified a host factor critical for N- glycosylation. We will test the hypothesis that the high speed with which RTA binds the ribosome together with its interaction with the host factors that facilitate translocation contribute to the cytotoxicity of ricin. We will carry out structure function analysis to identify residues that are critical for ribosome binding and examine the depurination activity and cytotoxicity of these mutants. We will determine if depletion of stal proteins in human cells will affect depurination activity and cytotoxicity of RTA. We will screen a high density peptide array library to identify peptide inhibitors of the ribosome docking event. We will determine how genes identified from the genetic screen in yeast mediate RTA toxicity in mammalian cells to identify potential therapeutic targets. These discoveries will impact our understanding ricin toxicity and will be critical for development of countermeasures with post-exposure potential. PUBLIC HEALTH RELEVANCE: Ricin intoxication causes serious and sometimes fatal effects. A critical gap exists in our knowledge of the mechanism of action of ricin, a potential weapon of bioterrorism. We propose to understand how ricin damages ribosomes and kills cells to develop antidotes. This research will provide a scientific basis for the development of remedies against ricin intoxication and will aid in the design of more effective therapeutic immunotoxins.